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The relationship between the insecticide dichloro-diphenyl-trichloroethane and chloroquine in Plasmodium falciparum resistance

Makowa, Hazel Beverly (2012-03)

Thesis (MSc)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: Dichloro-diphenyl-trichloroethane (DDT) was extensively used in agriculture pest control and
is still used for indoor residual spraying to control malaria. The lipophylicity of DDT and its
breakdown product dichloro-diphenyl-dichloroethylene (DDE) dictates that they associate
with membranes, lipids and hydrophobic proteins in the biological environment. Their poor
degradable nature causes DDT and DDE to persist for decades in the environment and in
individuals who are or were in contact with the pesticide. In many countries the synchronised
resistance of the mosquito vector to insecticides and the malaria parasite towards antimalarial
drugs led to a drastic rise in malaria cases and to malaria epidemics. This study assesses the
influence of low level exposure of DDT and DDE on chloroquine (CQ) resistance of the dire
human malaria parasite, Plasmodium falciparum.
The in vitro activity of p,p’-DDT and p,p’-DDE towards blood stages of chloroquine sensitive
(CQS) P. falciparum D10 and chloroquine resistant (CQR) P. falciparum Dd2 was
determined using two complementary in vitro assays (Malstat and SYBR Green 1). The 50%
inhibition concentrations (IC50s) of p,p’-DDT and p,p’-DDE were found to be ±14 to 38 μM
(5-12 μg/mL) and highly similar towards CQS and CQR P. falciparum strains. This result
indicated that the proteins involved in CQ resistance have no effect on the activity of the
insecticide DDT and it breakdown product DDE.
In order to assess the influence of DDT and DDE on CQ activity, in vitro fixed ratio drug
combination assays were performed, as well as isobologram analysis. We found that CQ
works in synergy with p,p’-DDT and p,p’-DDE against CQS P. falciparum D10. However,
both p,p’-DDT and p,p’-DDE were antagonistic toward CQ activity in CQR P. falciparum
Dd2. This indicated that p,p’-DDT and p,p’-DDE do have an effect on CQ resistance or on
the action of CQ via a target other than hemozoin polymerization. The observation of
reciprocal synergism of p,p’-DDT and p,p’-DDE with CQ against CQS D10 and antagonism
against CQR Dd2 strain is highly significant and strongly indicates selection of CQ resistant
strains in the presence of p,p’-DDT and p,p’-DDE. People who have low levels of circulating
DDE and/or DDT could be at a high risk of contracting CQR malaria. However, medium term
(nine days) DDE exposure of CQS P. falciparum D10 did not induce resistance, as no
significant change in activity of CQ, p,p’-DDT and p,p’-DDE towards blood stages the CQS
strain was observed. This exposure was, however, shorter than expected for a malaria
infection and would be addressed in future studies.
From our results on the interaction of CQ with p,p’-DDT and p,p’-DDE, it was important to
assess the residual DDT and DDE variable and how much of residual p,p’-DDT and/or p,p’-
DDE would enter into or remain in the different compartments (the RPMI media, erythrocytes
and infected erythrocytes) over time. In combination with liquid-liquid extraction, we
developed a sensitive GC-MS analyses method and a novel HPLC-UV analysis method for
measuring DDT and DDE levels in malaria culturing blood and media. Whilst the HPLC-UV
method was relatively cheaper, faster, and effective in determining high DDT and DDE
concentrations, the optimised GC-MS method proved to be effective in detecting levels as
low as 78 pg/mL (ppt) DDE and 7.8 ng/mL (ppb) DDT in biological media. Using both the
HPLC and GC-MS methods we observed that malaria parasites influence distribution of the
compounds between the erythrocytic and media fractions. P. falciparum D10 infection at
±10% parasitemia lead to must faster equilibration (less than 8 hours) between compartments.
Equimolar distribution of p,p’-DDE was observed, but the parasites lead to trapping of the
largest fraction of p,p’-DDT in the erythrocyte compartment. These results indicate that a
substantial amount would reach the intra-erythrocytic parasite and could influence the
parasite directly, possibly leading to either synergistic or antagonistic drug interactions.
This study is the first to illustrate the “good and bad” of the insecticide DDT in terms of CQ
resistance and sensitivity toward the human malaria parasite P. falciparum. These results will
hopefully have an important influence on how future policies on malaria control and
treatment particularly in endemic areas will be addressed and could also have an impact on
the anti-malarial drug discovery approach.